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Noncovalent Interactions
A Weak Attractive Interaction between
Organic Fluorine and an Amide Group
A combination of a chemical doublemutant cycle and a linear free energy
relationship has demonstrated that a
weak attractive interaction ( 0.8 to
1.5 kJ mol 1) exists between an organic
fluorine substituent and the face of an
amide functional group (see picture). This
study supports recent results that have
suggested that such an attraction may be
operative in enzyme–inhibitor interactions.
F. Hof, D. M. Scofield, W. B. Schweizer,
F. Diederich*
5056 – 5059
Keywords: amides · fluorine ·
molecular recognition ·
noncovalent interactions
2004 – 43/38
WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Communications
Noncovalent Interactions
A Weak Attractive Interaction between Organic
Fluorine and an Amide Group**
Fraser Hof, Denise M. Scofield, W. Bernd Schweizer,
and Franois Diederich*
Questions regarding the nature and strength of noncovalent
interactions formed by organic fluorine atoms are increasingly being addressed and debated in the literature.[1] We have
been exploring noncovalent interactions of fluorine by
carrying out a systematic fluorine scan[2] at the active site of
thrombin.[3] While exploring the hydrophobic D pocket of this
serine protease, we noticed that the potency of a closely
related family of fluorinated inhibitors was strongly influenced by the position of the fluorine atom (Figure 1).[3a] The
4-fluorobenzyl derivative ( )-4 exhibits fivefold better
inhibition than any other member of the family. The X-ray
structure of the (+)-4–enzyme complex showed two close
contacts between the fluorine atom and the CaH atom as
well as the carbonyl C atom of Asn98 (Figure 1 b). Subsequent searches in the Cambridge Structural Database and
RCSB Protein Data Bank provided numerous examples of
similar sub van der Waals contacts between organic fluorine
atoms and carbonyl carbon atoms in chemical and biological
samples. These interactions have a characteristic geometry:
the fluorine atom tends to reside orthogonally above the
pseudotrigonal axis of the carbonyl group, and the CF bond
approaches the plane of the carbonyl group from an angle
between 1008 and, at very short F···C distances, 1408.[3a]
Herein we report the first model system to evaluate the
energetics of the proposed CF···amide interactions.
The distinct geometry of the orthogonal CF···amide
interaction presented an unusual challenge. We found an
answer in the “molecular torsion balance” derived by Wilcox
et al. from the Tr:ger base—a system designed for the
accurate measurement of edge-to-face aromatic–aromatic
interactions by the observation of a simple conformational
equilibrium.[4] We found through examination of existing
crystal structures[5] that appropriate substitution of the Tr:ger
base skeleton would provide the perpendicular arrangement
of functional groups required for our study (Scheme 1). The
Scheme 1. The torsion balance of Wilcox and co-workers. Modeling
studies predict both edge-to-face aromatic–aromatic interactions and
close contacts between substituents R1 and R2 for the folded state.
use of a trifluoromethyl group was required to approximate
the optimal CF···amide geometry. Based on the similarity of
the results obtained from database
searches for fluorine atoms attached to
sp2- and sp3-hybridized carbon atoms, we
anticipated that this substitution would
have a negligible effect on the energetics
of the proposed interaction.
The second challenge in characterizing fluorine–amide interactions is their
expected weakness. The relative Ki values for compounds ( )-1–( )-7 suggest
that the fluorine substitution provides
Figure 1. a) A family of inhibitors designed to probe the fluorophilicity of the D pocket at the
approximately 4 kJ mol1 of stabilizing
thrombin active site. b) X-ray structure showing close contacts between the fluorine atom of
energy. Hunter and co-workers have
inhibitor (+)-4 and the protein backbone of thrombin.
popularized chemical double-mutant
cycles for the measurement of very
small interaction energies in supramolecular
systems,
and
have
used this method to accurately
[*] Dr. F. Hof, D. M. Scofield, Dr. W. B. Schweizer, Prof. Dr. F. Diederich
measure
various
noncovalent
interactions as weak as
Laboratorium f1r Organische Chemie
1 kJ mol1.[6] The application of this strategy to the Wilcox
ETH-H5nggerberg, HCI
8093 Z1rich (Switzerland)
torsion balance is straightforward. The edge-to-face aroFax: (+ 41) 1-632-1109
matic–aromatic interaction is the primary force behind the
E-mail: [email protected]
folding of the molecule, and the effect of each substitution on
[**] F.H. thanks the Novartis Foundation and the Human Frontier
this primary force must be accounted for to determine the
Science Program for postdoctoral fellowship support. This research
incremental folding free enthalpy provided by the two
was supported by the ETH Research Council and F. Hoffmannappended functional groups. A double-mutant cycle
La Roche Ltd.
(Scheme 2) can be used to determine the influence of each
Supporting information for this article (synthesis and characterappended group on the edge-to-face interaction independization of compounds ( )-8–( )-10 and ( )-13 as well as
ently from their interaction with each other. When the folding
complete error analysis of the physical data) is available on the
energies of all four molecules in the double-mutant cycle have
WWW under http://www.angewandte.org or from the author.
5056
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/anie.200460781
Angew. Chem. Int. Ed. 2004, 43, 5056 –5059
Angewandte
Chemie
be determined with well-defined estimations of error. The
singlets corresponding to the methyl resonances of each
folded state are resolved at 300 MHz, and the identity of each
conformer is easily determined by analogy with related
compounds (Figure 2).[4] A summary of the folding energies
Scheme 2. Double-mutant cycle for determining the magnitude of the
attraction between orthogonally oriented CF3 and NHCOCH3 groups.
Figure 2. 1H NMR spectra (methyl region, C6D6, c = 10 mm) of: a) ( )8, b) ( )-9, c) ( )-10, d) ( )-11. The sensitivity of the acetamide
COCH3 resonance to the proximity of the trifluoromethyl group in the
folded state is highlighted.
been determined, the interaction energy of the two appended
functional groups (DGCF3···NHCOCH3) is given by Equation (1).
DGCF3 NHCOCH3 ¼ DGðÞ-8 DGðÞ-9 DGðÞ-10 þ DGðÞ-11
ð1Þ
The four molecules of interest were synthesized as shown
in Scheme 3. The nitrocarboxylic acid intermediate ( )-12,
prepared in thirteen steps using literature methods,[7] was
esterified with 4-(trifluoromethyl)phenol and phenol to give
( )-13 and ( )-14, respectively.
for compounds ( )-8–( )-11 in three different solvents is
given in Table 1. The application of the double-mutant cycle
in the form of Equation (1) provides the incremental free
enthalpy for the interaction of the two appended functional
groups. The interaction is weak, but measurable, in CDCl3
(1.05 0.25 kJ mol1) and C6D6 (0.85 0.25 kJ mol1),
while the value is essentially zero in CD3OD (0.10 0.25 kJ mol1).
Table 1: Folding free enthalpies for compounds ( )-8–( )-11.
Compound
( )-8
( )-9
( )-10
( )-11
Scheme 3. Synthesis of ( )-8–( )-11. a) Et3N, BOP, 4-(trifluoromethyl)phenol or phenol, CH2Cl2, RT, 71–97 %. b) H2 (atm), RaNi, EtOAc/
EtOH, RT, 90–100 %. c) Ac2O, CH2Cl2, RT, 20–72 %. d) 1. HCl, NaNO2,
5 8C; 2. H3PO2, 5 8C, 25–63 %. BOP = Benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate, RaNi = Raney Nickel.
Reduction of the nitro group with H2/RaNi gives the
corresponding anilines. In divergent steps, each aniline is then
acetylated to yield target compounds ( )-8 and ( )-10, or
converted into its corresponding diazonium salt, which is in
turn reduced with H3PO2 to afford compounds ( )-9 and
( )-11.
The interconversion between the two conformations of
each molecule is slow on the NMR timescale (Ea = 65–
75 kJ mol1).[4a, 8] Direct integration of the 1H and/or 19F resonances for each conformer allows the folding equilibrium to
Angew. Chem. Int. Ed. 2004, 43, 5056 –5059
DG [ 0.12 kJ mol1][a]
CDCl3
C6D6
CD3OD
2.46
1.95
0.45
1.00
2.88
2.37
1.72
1.31
3.78
3.31
1.46
1.84
[a] Determined by integration of signals in the NMR spectra of 10 mm
solutions at 298 K. For a complete error analysis, see the Supporting
Information.
Two main conditions must be satisfied for a doublemutant cycle to be valid. Firstly, the core must not undergo
major structural rearrangements between different members
of the cycle. With few degrees of freedom, the scaffold of
Wilcox and co-workers derived from the Tr:ger base is
exceptionally well organized. To determine the effect of
trifluoromethyl substitution on the folded structure we solved
the crystal structure of synthetic precursor ( )-13
(Figure 3).[9] Comparison with several existing crystal structures[5] of related torsion-balance molecules shows there is
little deviation in the folded edge-to-face geometry throughout the series. The crystal structure of precursor ( )-13 also
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2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5057
Communications
pound ( )-11 to ( )-9) causes a much larger favorable
secondary effect (D(DG) = 1.5 kJ mol1 in C6D6). This
difference arises from the acidification of the neighboring
ortho proton, which increases the strength of the CH–
p interaction at the heart of the folded conformation.
To get a better understanding of the effect of the
trifluoromethyl group on the core edge-to-face interaction
we synthesized a series of compounds in which the “face”
component bears different substituents. The folding equilibrium constant of each was determined by using the same
method as for compounds ( )-8–( )-11, and the entire series
was examined using a linear free energy relationship between
the folding free enthalpy and the Hammett constants smeta
(Figure 4).[10] The correlation is excellent (R2 > 0.98), thus
Figure 3. ORTEP plot with ellipsoids at 50 % level showing the folded
state of ( )-13. Distances between F(32) and the atoms of the nitro
group are given in H.
displays the desired perpendicular approach of the appended
functional groups (in this case a trifluoromethyl and a nitro
group). The trifluoromethyl group experiences no attraction
to the adjacent nitro group (see below), and accordingly the
close interatomic contacts involving F(32) are greater than
the sum of van der Waals radii (Figure 3). Modeling studies
on ( )-8 suggest that the minute (< 0.3 G) vertical adjustment required to bring the fluorine atom close to the plane of
the acetamide group can be accommodated, despite the
rigidity of the scaffold. However, the modeling study also
shows that for ( )-8 the fluorine atom closest to the
acetamide group may not reach its ideal position above the
pseudotrigonal axis of the carbonyl unit. Instead, it may lie
somewhere above the bond connecting the nitrogen atom to
the carbonyl carbon atom, which suggests there is a geometric
factor that can account for the slightly weaker than expected
value measured for the fluorine–amide interaction.
In the absence of an X-ray structure of ( )-8, we turned
to NMR studies to provide evidence for the proximity of the
trifluoromethyl and acetamide groups in solution. The difference in the chemical shifts observed for the acetamide CH3
group in the folded and unfolded states is larger for ( )-8
(Dd = 0.30 ppm) than for control compound ( )-10 (Dd =
0.14 ppm), which suggests that the acetamide residue experiences decreased shielding in the folded state of ( )-8
because of the nearby fluorine atoms (Figure 2).
The validity of a double-mutant cycle also rests on the
linear additivity of the secondary energy perturbations arising
from each functional group mutation. A closer analysis of the
data in Table 1 shows that the addition of an acetamide group
to the “face” component of the primary edge-to-face interaction (from compound ( )-11 to ( )-10) causes a small
unfavorable change in the folding free enthalpy (D(DG) =
+ 0.4 kJ mol1 in C6D6). In comparison, the addition of a
trifluoromethyl group to the “edge” component (from com-
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2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 4. A linear free energy relationship showing substituent effects
for edge-to-face interactions in a family of trifluoromethyl-substituted
molecules.
showing that the primary CH–p interaction follows a wellbehaved continuum that depends on the electronic properties
of the “face” component. The sole significant departure from
this trend arises for the acetamide-substituted compound
( )-8.[11] This compound is more folded by 1.5 0.7 kJ mol1
than substituent effects would predict—this value is similar to
that obtained from the double-mutant cycle analysis.
What is the origin of the attraction between the appended
trifluoromethyl and acetamide groups? It is unlikely that the
amide NH group can be appropriately oriented to donate a
hydrogen bond to the fluorine atoms, and the lack of any
attraction for the more acidic phenol compound (Figure 4)
confirms that this mode of weak hydrogen bonding does not
stabilize the folded state.[1a] It is possible that the model
suggested by our previous database findings, in which the
electronegative end of the carbon–fluorine dipole interacts
favorably with the electropositive carbonyl carbon atom, is
operative.[3a] This dipolar electrostatic model accounts for the
absence of any measurable attraction in the polar solvent
CD3OD. Alternatively, one can consider the approach of the
unpolarizable, partially negative surface of the trifluoro-
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Angew. Chem. Int. Ed. 2004, 43, 5056 –5059
Angewandte
Chemie
methyl group[12] close to the polarizable delocalized p cloud of
the amide group. Attraction arises in this model from chargeinduced dipole and dipole-induced dipole interactions. Arguments against such a model are indicated by a recent study in
which a trifluoromethyl group is not attracted by the face of
an aryl ring, regardless of the electrostatic potential of the
ring.[13] In the absence of structural information for compound
( )-8, it is impossible to conclude whether an atom-centered
model or a functional-group-scale model of this attraction is
more appropriate. We hope that further studies using different fluorine-containing functional groups and different carbonyl functional groups will shed light on this issue.
In summary, we have presented the first efforts to quantify
the interactions of organic fluorine with the face of an amide
functional group. A novel combination of the torsion balance
developed by Wilcox and co-workers with two distinct
physical methods—a double-mutant cycle and a linear free
energy relationship—provided the initial measurement of an
attractive interaction with a value of 0.8–1.5 kJ mol1 in
nonpolar solvents. The effects of fluorination on the hydrophobicity, distribution, metabolism, and pharmacokinetics of
potential drug molecules are increasingly well understood.[14]
Model studies such as those presented here help clarify the
role played by fluorine atoms in altering the binding affinity
and selectivity[3, 14d] of enzyme inhibitors.
[7]
[8]
[9]
[10]
Received: May 25, 2004
.
Keywords: amides · fluorine · molecular recognition ·
noncovalent interactions
[11]
[12]
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[5] The X-ray structures of two related compounds can be found in
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[6] a) H. Adams, F. J. Carver, C. A. Hunter, J. C. Morales, E. M.
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E.-I. Kim, PhD thesis, University of Pittsburgh (USA), 1996.
S. Paliwal, PhD thesis, University of Pittsburgh (USA), 1996.
Crystals of ( )-13 were grown by vapor diffusion of pentane
into a solution of ( )-13 in EtOAc: yellow prism, 0.4 S 0.1 S
0.1 mm; orthorhombic, space group F2dd; a = 10.36, b = 25.37,
c = 38.17 G, V = 10 030 G3 ; 1calcd = 1.445 Mg m3 ; 2qmax = 60.068;
MoKa radiation, l = 0.71073 G; T = 152 K; 7164 independent of
7301 measured reflections Rint = 0.032, no absorption correction
applied (m = 0.113 mm1); structure solution using SIR97 (A.
Altomare, M. C. Burla, M. Camalli, G. L. Cascarano, C.
Giacovazzo, A. Guagliardi, A. G. G. Moliterni, R. Spagna, J.
Appl. Crystallogr. 1999, 32, 115 – 119); 5402 reflections with I >
3s(I) refined on j F2 j using SHELXL-97 (G. M. Sheldrick,
SHELXL97, Program for the Refinement of Crystal Structures,
University of G:ttingen, G:ttingen (Germany), 1997); D/smax =
0.299, D1max = 0.734 e G3, D1min = 0.482 e G3 ; 449 parameters,
all hydrogen atom parameters refined;, R(all) = 0.0953, R(gt) =
0.0653, wR(ref) = 0.1920, wR(gt) = 0.1603. CCDC-239177 contains the supplementary crystallographic data for this paper and
is available free of charge from the Cambridge Crystallographic
Data Centre (CCDC, 12 Union Road, Cambridge CB2 1EZ
(UK); Tel.: (+ 44) 1223-336-408, Fax: (+ 44) 1223-336-033, Email: [email protected]).
a) The linear free energy relationships of other edge-to-face
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Hunter, D. J. Livingstone, J. F. McCabe, E. M. Seward, Chem.
Eur. J. 2002, 8, 2847 – 2859; b) all values for the Hammett
constants smeta were obtained from: D. H. McDaniel, H. C.
Brown, J. Org. Chem. 1958, 23, 420 – 427.
The trifluoromethyl group should experience some repulsion
when brought close to the large iodine atom. This effect is
evident in the slightly under-folded state of the iodo compound.
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H. Adams, S. L. Cockroft, C. Guardigli, C. A. Hunter, K. R.
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2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5059